U.S. patent number 5,707,986 [Application Number 08/209,473] was granted by the patent office on 1998-01-13 for angiographic method using green porphyrins in primate eyes.
Invention is credited to Evangelos S. Gragoudas, Joan W. Miller, Lucy H.Y. Young.
United States Patent |
5,707,986 |
Miller , et al. |
January 13, 1998 |
Angiographic method using green porphyrins in primate eyes
Abstract
An angiographic method to observe the condition of blood
vessels, including neovasculature in the eyes of living primates
using green porphyrins and light at a wavelength of 550-700 nm to
effect fluorescence is disclosed.
Inventors: |
Miller; Joan W. (Boston,
MA), Young; Lucy H.Y. (Boston, MA), Gragoudas; Evangelos
S. (Boston, MA) |
Family
ID: |
22778888 |
Appl.
No.: |
08/209,473 |
Filed: |
March 14, 1994 |
Current U.S.
Class: |
514/185; 514/410;
514/912 |
Current CPC
Class: |
A61K
9/0019 (20130101); A61K 9/1275 (20130101); A61K
31/409 (20130101); A61K 31/555 (20130101); A61K
49/0036 (20130101); A61K 49/0084 (20130101); A61K
41/0071 (20130101); Y10S 514/912 (20130101) |
Current International
Class: |
A61K
41/00 (20060101); A61K 49/00 (20060101); A61K
031/40 (); A61K 047/42 () |
Field of
Search: |
;514/185,410,912 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Schmidt et al., "Photosensitizing Potency of Benzoporphyrin
Derivative (BPD) Associated with Human Low Density Lipoprotein
(LDL)", IOVS(1992) 33:1253 Abstract 2802. .
Lin et al., "Measurement of BPD Photosensitizer Kinetics in Retinal
and Choroidal Vessels by Fluorescence Imaging", IOVS (1933) 34:1168
Abstract 2293. .
Lin et al., "Photodynamic Closure of Choroidal Vessels Using
Benzoporphyrin Derivative", IOVS (1993) 34:1303 Abstract 2953.
.
Schmidt-Erfurth et al., "Photothrombosis of Ocular
Neovascularization Using Benzoporphyrin (BPD)", IOVS (1993) 34:1303
Abstracts 2956. .
Haimovici et al., "Localization of Benzoporphyrin Derivative
Monoacid in the Rabbit Eye", IOVS (1993) 34:1303 Abstracts 2955.
.
Walsh et al., "Photodynamic Therapy of Experimental Choroidal
Neovascularization Using Benzoporphyrin Derivative Monoacid", IOVS
(1993) 34:1303 Abstracts 2954. .
Moulton et al., "Response of Retinal and Choroidal Vessels to
Photodynamic Therapy Using Benzoporphyrin Derivative Monoacid",
IOVS (1993) 34:1169 Abstract 58. .
Gonzales et al., "Photodynamic Therapy of Pigmented Choroidal
Melanomas", IOVS (1993) 34:891 Abstract 949. .
Schmidt-Erfurth et al., "Photodynamic Therapy of Experimental
Choroidal Melanoma Using Lipoprotein-delivered Benzoporphyrin",
Ophthalmology (1994) 101:89-99. .
Pandey et al., "Efficient Synthesis of New Classes of
Regiochemically Pure Benzoporphyrin Derivatives", Bioorganic &
Medicinal Chemistry Letters (3)12:2615-2618 (1993)..
|
Primary Examiner: Fay; Zohreh
Attorney, Agent or Firm: Morrison & Foerster LLP
Claims
We claim:
1. An angiographic method to observe the condition of blood
vessels, including neovasculature in the eye of an intact living
primate, which method comprises:
a. administering to said primate comprising at least one eye for
which said observation is desired and containing said
neovasculature an amount of green porphyrin which will provide an
observable amount of green porphyrin in the blood vessels and
neovasculature of said eye;
b. permitting sufficient time to elapse so that an observable
amount of said green porphyrin resides in the blood vessels and
neovasculature of the eye; and
c. applying light of a wavelength in the range of about 550-700 nm
to the eye so as to effect fluorescence of the green porphyrin;
and
d. detecting said fluorescence with a suitable camera.
2. The method of claim 1 wherein said green porphyrin is a BPD.
3. The method of claim 2 wherein said BPD is BPD-MA.
4. The method of claim 1 wherein said green porphyrin is complexed
with low-density lipoprotein.
5. The method of claim 1 wherein said green porphyrin is contained
in a liposomal preparation.
6. The method of claim 3 wherein said BPD-MA is contained in a
liposomal preparation.
Description
TECHNICAL FIELD
The invention is in the field of photodynamic therapy, specifically
related to ocular conditions. More particularly, the invention
concerns the use of green porphyrins in photodynamic therapeutic
treatment of pigmented tumors and conditions characterized by
unwanted neovasculature in the eye. Green porphyrins are also
useful as dyes in ocular angiography.
BACKGROUND ART
Choroidal neovascularization leads to hemorrhage and fibrosis, with
resultant visual loss in a number of eye diseases, including
macular degeneration, ocular histoplasmosis syndrome, myopia, and
inflammatory diseases. Age-related macular degeneration is the
leading cause of new blindness in the elderly, and choroidal
neovascularization is responsible for 80% of the severe visual loss
in patients with this diseases. Although the natural history of the
disease is eventual quiescence and regression of the
neovascularization process, this usually occurs at the cost of
sub-retinal fibrosis and vision loss.
Current treatment relies on occlusion of the blood vessels using
laser photocoagulation. However, such treatment requires thermal
destruction of the neovascular tissue, and is accompanied by
full-thickness retinal damage, as well as damage to medium and
large choroidal vessels. Further, the subject is left with an
atrophic scar and visual scotoma. Moreover, recurrences are common,
and visual prognosis is poor.
Developing strategies have sought more selective closure of the
blood vessels to preserve the overlying neurosensory retina. One
such strategy is photodynamic therapy, which relies on low
intensity light exposure of photosensitized tissues to produce
photochemical effects. Photosensitizing dyes are preferentially
retained in tumors and neovascular tissue, which allows for
selective treatment of the pathologic tissue. As a result of the
invention, PDT may be used to cause vascular occlusion in tumors by
damaging endothelial cells, as well as a direct cytotoxic effect on
tumor cells.
Photodynamic therapy of conditions in the eye characterized by
neovascularization has been attempted over the past several decades
using the conventional porphyrin derivatives such as
hematoporphyrin derivative and Photofrin porfimer sodium. Problems
have been encountered in this context due to interference from eye
pigments. In addition, phthalocyanine has been used in photodynamic
treatment.
A newer photosensitizer, a member of the group designated "green
porphyrins", is in the class of compounds called benzoporphyrin
derivatives (BPD). This photosensitizer has also been tested to
some extent in connection with ocular conditions. For example,
Schmidt, U. et al. described experiments using BPD for the
treatment of Greene melanoma (a nonpigmented tumor) implanted into
rabbit eyes and achieved necrosis in this context (IOVS (1992)
33:1253 Abstract 2802). Lin, C. P. et al. describe the measurement
of kinetics and distribution in retinal and choroidal vessels by
fluorescence imaging using a 458 nm line from an argon-ion laser to
excite BPD (IOVS (1993) 34:1168 Abstract 2293). In addition, Lin,
S. C. et al. described photodynamic closure of choroidal vessels
using BPD in IOVS (1993) 34:1303 Abstract 2953.
The present applicants have described treating choroidal
neovascularization using BPD in several abstracts published 15 Mar.
1993 and incorporated herein by reference. These abstracts include
Schmidt-Erfurth, U. et al. "Photothrombosis of Ocular
Neovascularization Using BPD"; Haimovici, R. et al. "Localization
of Benzoporphyrin Derivative Monoacid in the Rabbit Eye"; and
Walsh, A. W. et al. "Photodynamic Therapy of Experimental Choroidal
Neovascularization Using BPD-MA." All of the foregoing are
published in IOVS (1993) 34:1303 as abstracts 2956, 2955 and 2954,
and Moulton, R. S. et al. "Response of Retinal and Choroidal
Vessels to Photodynamic Therapy Using Benzoporphyrin Derivative
Monoacid", IOVS (1993) 34:1169 Abstract 58.
The green porphyrins offer advantages in their selectivity for
neovasculature and in their ability to effect photodynamically
mediated destruction of nonpigmented tumors of the eye. Gonzales et
al. reported photodynamic therapy of pigmented melanomas in the eye
using phthalocyanine as a photoactive compound (Gonzales, Y. H. et
al. (IOVS (1993) 34:891 Abstract 949). The use of BPD-MA in
photodynamic treatment of the nonpigmented Greene hamster melanoma
in rabbit eyes was also described by Schmidt-Erfurth, U. et al. in
Ophthalmology (1994) 101:89-99. This report was presented in part
at the ARVO annual meeting in Sarasota, Fla., May 1992.
DISCLOSURE OF THE INVENTION
The invention is directed to diagnosis and treatment of certain
conditions of the eye using photodynamic methods and employing
green porphyrins as the photoactive compounds. The green porphyrins
of the invention are described in U.S. Pat. Nos. 4,883,790;
4,920,143; 5,095,030; and 5,171,749, the entire contents of which
are incorporated herein by reference. These materials offer
advantages of selectivity and effectiveness when employed in
protocols directed to the destruction of unwanted neovasculature
and pigmented tumors. They are also particularly effective as
visualizing agents in angiography of ocular blood vessels. The
visualization of the compounds is enhanced by their ability to
fluoresce.
Accordingly, in one aspect, the invention is directed to a method
to treat conditions of the eye characterized by unwanted
neovasculature, which method comprises administering to a subject
in need of such treatment an amount of a green porphyrin that will
localize in said neovasculature; and irradiating the neovasculature
with light absorbed by the green porphyrin.
In another aspect, the invention is directed to a method to treat
pigmented tumors in the eye, which method comprises administering
to a subject in need of such treatment an amount of a green
porphyrin that will localize in said tumor; and irradiating the
tumor with light absorbed by the green porphyrin.
In still another aspect, the invention is directed to a method to
observe the condition of blood vessels in the eye, which method
comprises administering to a subject comprising at least one eye
for which said observation is desired an amount of green porphyrin
which will provide an observable amount of green porphyrin in the
blood vessels of said eye, permitting sufficient time to elapse so
that an observable amount of said green porphyrin resides in the
eyes; and observing the blood vessels in which the green porphyrin
resides.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows preferred forms of the green porphyrins useful in the
methods of the invention.
MODES OF CARRYING OUT THE INVENTION
The methods of the claimed invention administering to a subject a
green porphyrin, which is in the class of compounds called
benzoporphyrin derivatives (BPD). A BPD is a synthetic chlorin-like
porphyrin with a number of structural analogues, as shown in FIG.
1.
Preferably, the green porphyrin is benzoporphyrin derivative
mono-acid ring A (BPD-MA), which absorbs light at about 692 nm
wavelength with improved tissue penetration properties. BPD-MA is
lipophilic, a potent photosensitizer, and appears to be phototoxic
to neovascular tissues and tumors.
In a preferred embodiment, the green porphyrin is prepared as a
liposomal preparation or is coupled to a ligand that binds to a
specific surface component of the neovasculature to improve even
further its effectiveness as a photosensitizer. Preferably, the
ligand comprises an antibody or an immunologically reactive
fragment thereof. The capacity for selective localization of a
green porphyrin can also be improved by coupling to a carrier
molecule that potentially delivers higher concentrations of the
green porphyrin to the target tissue.
A carrier that is appropriate for clinical use is human low-density
lipoprotein (LDL). Human LDL is a physiologic serum protein
metabolized by cells via uptake by high affinity receptors. LDL
exhibits desirable characteristics as a selective carrier because
LDL metabolism is increased in tumor cells. Neoplastic tissues and
neovascularization have been shown to have increased numbers of LDL
receptors. Further, by increasing the partitioning of the green
porphyrin into the lipoprotein phase of the blood, it appears to be
delivered more efficiently to the target tissue.
Because of its lipophilicity and negative charge, green porphyrins
strongly interact with lipoproteins. Most preferably, the green
porphyrin is complexed with low density lipoprotein (LDL).
When injected intravenously, BPD-MA is cleared from the bloodstream
with a half-life of about 10-30 minutes, with the highest tissue
levels being reached in about three hours after administration by
injection and declining rapidly in the first 24 hours. BPD-MA is
cleared primarily via bile and feces (60%), with only 4% being
cleared via the kidneys and urine. Thus, skin photosensitivity
occurs with BPD-MA only transiently, with minimal reactivity after
24 hours in in vivo models.
The green porphyrin can be administered in any of a wide variety of
ways, for example, orally, parenterally, or rectally. Parenteral
administration, such as intravenous, intramuscular, or
subcutaneous, is preferred. Intravenous injection is especially
preferred.
The dose of green porphyrin can vary widely depending on the tissue
to be treated; the physical delivery system in which it is carried,
such as in the form of liposomes, or whether it is coupled to a
target-specific ligand, such as an antibody or an immunologically
active fragment.
It should be noted that the various parameters used for effective,
selective photodynamic therapy in the invention are interrelated.
Therefore, the dose should also be adjusted with respect to other
parameters, for example, fluence, irradiance, duration of the light
used in photodynamic therapy, and the time interval between
administration of the dose and the therapeutic irradiation. All of
these parameters should be adjusted to produce significant damage
to neovascular or tumor tissue without significant damage to the
surrounding tissue or, on the other hand, to enable the observation
of blood vessels in the eye without significant damage to the
surrounding tissue. Typically, the dose of green porphyrin used is
within the range of from about 0.1 to about 20 mg/kg, preferably
from about 0.15-2.0 mg/kg, and even more preferably from about 0.25
to about 0.75 mg/kg.
Specifically, as the green porphyrin dose is reduced from about 2
to about 1 mg/kg, the fluence required to close choroidal
neovascular tissue tends to increase, for example, from about 50 to
about 100 Joules/cm.sup.2.
After the photosensitizing green porphyrin has been administered,
the choroidal neovascular tissue or tumor being treated or observed
in the eye is irradiated at the wavelength of maximum absorbance of
the green porphyrin, usually between about 550 and 695 nm. A
wavelength in this range is especially preferred for enhanced
penetration into bodily tissues.
As a result of being irradiated, the green porphyrin in its triplet
state is thought to interact with oxygen and other compounds to
form reactive intermediates, such as singlet oxygen, which can
cause disruption of cellular structures. Possible cellular targets
include the cell membrane, mitochondria, lysosomal membranes, and
the nucleus. Evidence from tumor and neovascular models indicates
that occlusion of the vasculature is a major mechanism of
photodynamic therapy, which occurs by damage to endothelial cells,
with subsequent platelet adhesion, degranulation, and thrombus
formation.
The fluence during the irradiating treatment can vary widely,
depending on type of tissue, depth of target tissue, and the amount
of overlying fluid or blood, but preferably varies from about
50-200 Joules/cm.sup.2.
The irradiance typically varies from about 150-900 mW/cm.sup.2,
with the range between about 150-600 mW/cm .sup.2 being preferred.
However, the use of higher irradiances may be selected as effective
and having the advantage of shortening treatment times.
The optimum time following green porphyrin administration until
light treatment can vary widely depending on the mode of
administration; the form of administration, such as in the form of
liposomes or as a complex with LDL; and the type of target tissue.
As a specific example, a time interval of 1-20 minutes is often
appropriate for retinal neovascular tissue, about 120 minutes is
allowed for choroidal neovascular tissue, and up to about three
hours may be allowed for tumors. Thus, effective vascular closure
generally occurs at times in the range of about one minute to about
three hours following administration of the green porphyrin.
The time of light irradiation after administration of the green
porphyrin may be important as one way of maximizing the selectivity
of the treatment, thus minimizing damage to structures other than
the target tissues. For a primate, it is believed that the green
porphyrin begins to reach the retinal vasculature by about 7-15
seconds following administration. Typically, the green porphyrin
persists for a period of about 5-15 minutes, depending on the dose
given. Treatment within the first five minutes following
administration of the green porphyrin should generally be avoided
to prevent undue damage to retinal vessels still containing
relatively high concentrations of the green porphyrin.
Clinical examination and fundus photography typically reveal no
color change immediately following photodynamic therapy, although a
mild retinal whitening occurs in some cases after about 24 hours.
Closure of choroidal neovascularization, however, is preferably
confirmed histologically by the observation of damage to
endothelial cells. Vacuolated cytoplasm and abnormal nuclei can
become apparent as early as 1-2 hours following photodynamic
therapy, with disruption of neovascular tissue typically becoming
more apparent by about 24 hours after light treatment. Associated
damage to the retinal pigment epithelium (RPE), pyknotic nuclei in
the outer nuclear layer, and loss of photoreceptors may also be
observed. However, the inner retina usually appears relatively
undamaged, as shown by control studies using photodynamic therapy
with BPD-MA on a normal retina and choroid showing no damage to
large choroidal and retinal vessels.
Closure can usually be observed angiographically by about 40
seconds to a minute in the early frames by hypofluorescence in the
treated areas. During the later angiographic frames, a corona of
hyperfluorescence begins to appear and then fills the treated area,
possibly representing leakage from the adjacent choriocapillaris
through damaged retinal pigment epithelium in the treated area.
Large retinal vessels in the treated area perfuse following
photodynamic therapy, but tend to demonstrate late staining.
Minimal retinal damage is generally found on histopathologic
correlation and is dependent on the fluence and the time interval
after irradiation that the green porphyrin is administered.
Histopathologic examination usually reveals vessel remnants in the
area of choroidal neovascular tissue, but the retinal vessels
typically appear normal. Further, there is no indication of
systemic toxicity, and cutaneous photosensitization does not appear
to develop.
As a result of the invention, photodynamic therapy can be used more
selectively, relying on the low intensity light exposure of green
porphyrins that have become localized within vascular tissue.
Complications, suck as serous detachment and hemorrhage, are not
noted with the invention. Thus, photodynamic therapy with a green
porphyrin appears to have broad application to clinical
ophthalmology in treating such diseases as age-related macular
degeneration, neovascular glaucoma, and persistent disc
neovascularization in diabetic retinopathy.
Photodynamic therapy using a green porphyrin can also be used
advantageously to treat not only nonpigmented tumors, but also
pigmented intraocular tumors such as pigmented choroidal melanomas,
and other pigmented tumors of the choroid, retina, iris or cornea.
The administered green porphyrin accumulates within the neoplastic
lesion and, upon localized light exposure at an appropriate
wavelength, the tumor tissue is thought to be irreversibly damaged
by the interaction with active molecular species like singlet
oxygen and other radicals induced by photoactivated dye molecules.
Maximal efficiency in tumor destruction, combined with minimal
irritation to adjacent physiological structures is a major benefit
in the treatment of intraocular malignancies, in particular,
choroidal tumors, which are in close contact with sensitive
neuronal and vascular structures.
Light microscopic evaluation of tumors that have not been treated
with photodynamic therapy show a mass of polygonal cells. Cell
nuclei are typically vesicular with prominent nucleoli, and mitotic
figures are scattered throughout the microscopic field. Tumors
typically exhibit many thin-walled blood vessels, which are
cylindrical in shape, are lined with intact endothelial cells, and
contain few erythrocytes.
After photodynamic therapy with a green porphyrin, however, tumor
blood vessels become dilated and densely packed with erythrocytes.
The tumor demonstrates clotted erythrocytes within the vascular
lumina. Several days later, homogeneous necrosis is typically
observable throughout the lesion. The tumor cells begin to exhibit
hyperchromatic nuclei and loss of cytoplasmic features. Further,
vascular structures show disintegration and endothelial cell
loss.
In treating tumors, the green porphyrin dosage administered may
vary widely depending on other parameters, as described above but,
preferably, is within the range of 0.5 to 3 mg/kg. The radiant
exposure can also range widely, depending on the pigmentation and
size of the tumor, but typically is in the range from about 60 to
about 2600 J/cm.sup.2. The level of light exposure is of particular
interest with respect to the time duration of the treatment.
In addition, green porphyrin can be used to observe the condition
of blood vessels in the eye, either alone or used in concert with
other dyes such as fluorescein or indocyanine green, as described
above to follow the progress of destruction choroidal neovascular
or tumor tissue. In such angiographic systems, a sufficient amount
of green porphyrin is administered to produce an observable
fluorescent emission when excited by light, preferably light having
a wavelength in the range of about 550-700 nm. Images are recorded
by illuminating the eye with light in the excitation wavelength
range and detecting the amount of fluorescent light emitted at the
emission wavelength. A preferred camera, which both emits and
receives light in the 550-700 nm range, is the TopCon 50VT camera
in the Ophthalmic Imaging System (Ophthalmic Imaging System Inc.,
221 Lathrop Way, Suite 1, Sacramento Calif.). An alternative camera
is the TopCon camera TRC 50IA connected to the TopCon Imagenet
System (TopCon America Corporation, 65 West Century Road, Taramus
N.J.).
In a preferred observation method of the invention, the green
porphyrin is administered, preferably by intravenous injection of a
bolus followed by a saline flush. The green porphyrin typically
reaches the retinal vasculature in about 7-15 seconds, and the
early angiographic frames are recorded after the first 20 seconds,
one every 3-5 seconds. Additional frames are taken periodically for
about two hours. A typical protocol might call for the image to be
recorded at about 40 seconds, then at 50 seconds, 60 seconds, two
minutes, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 60 minutes,
and two hours.
A camera is usually used to collect the emitted fluorescent light,
digitize the data, and store it for later depiction on a video
screen, as a hard paper copy, or in connection with some other
imaging system. While a film recording device may be used when
additional dyes such as fluorescein are being used in combination
with the green porphyrin, a CCD camera (video recording device) is
preferable as being able to capture emissions at higher
wavelengths, thus producing greater tissue penetration. As a
result, one can obtain more sophisticated information regarding the
pattern and extent of vascular structures in different ocular
tissue layers, giving the ability to detect the "leakiness" that is
characteristic of new or inflamed blood vessels. Further, it is
preferable to use a camera that is capable of providing the
excitation light, appropriately filtered to deliver only light of
the desired excitation wavelength range, and then to capture the
emitted, fluorescent light with a receiving device, appropriately
filtered to receive only light in the desired emission wavelength
range.
The following examples are to illustrate but not to limit the
invention.
EXAMPLE 1
Control of Experimental Choroidal Neovascularization Using PDT with
BPD-MA/LDL at Low Irradiance
Cynomolgus monkeys weighing 3-4 kg were anesthetized with an
intramuscular injection of ketamine hydrochloride (20 mg/kg),
diazepam (1 mg/kg), and atropine (0.125 mg/kg), with a supplement
of 5-6 mg/kg of ketamine hydrochloride as needed. For topical
anesthesia, proparacaine (0.5%) was used. The pupils were dilated
with 2.5% phenylephrine and 0.8% tropicamide.
Choroidal neovascularization was produced in the eyes of the
monkeys using a modification of the Ryan model, in which burns are
placed in the macula, causing breaks in Bruch's membrane, with a
Coherent Argon Dye Laser #920, Coherent Medical Laser, Palo Alto,
Calif. (Ohkuma, H. et al. Arch. Ophthalmol. (1983) 101:1102-1110;
Ryan, S. J. Arch Ophthalmol (1982) 100:1804-1809). Initially, a
power of 300-700 mW for 0.1 seconds was used to form spots of about
100 .mu.m, but improved rates of neovascularization were obtained
with 50.mu. spots formed using a power of about 300-450 mW for 0.1
second.
The resulting choroidal neovascularizations were observed by (1)
fundus photography (using a Canon Fundus CF-60Z camera, Lake
Success, Long Island, N.Y.); (2) by fluorescein angiography (for
example, by using about 0.1 ml/kg body weight of 10% sodium
fluorescein via saphenous vein injection); and (3) histologic
examination by light and electron microscopy.
Immediately before use, BPD-MA was dissolved in dimethyl sulfoxide
(Aldrich Chemical Co., Inc., Milwaukee, Wis.) at a concentration of
about 4 mg/ml. Dulbeccos phosphate buffered salt solution
(Meditech, Washington, D.C.) was then added to the stock to achieve
a final BPD concentration of 0.8 mg/ml. Human
low-density-lipoprotein (LDL) prepared from fresh frozen plasma was
added at a ratio of 1:2.5 mg BPD-MA:LDL. The green porphyrin dye
and dye solutions were protected from light at all times. After
mixing, the dye preparation was incubated at 37.degree. for 30
minutes prior to intravenous injection. The monkeys were then
injected intravenously via a leg vein with 1-2 mg/kg of the BPD-MA
complexed with LDL over a five-minute period, followed by a flush
of 3-5 cc of normal saline.
Following this intravenous injection, the eyes of the monkeys were
irradiated with 692 nm of light from an argon/dye laser (Coherent
920 Coherent Medical Laser, Palo Alto, Calif.), using a Coherent
LDS-20 slit lamp. The standard fiber was coupled to larger 400
.mu.m silica optical fiber (Coherent Medical Laser, Pal Alto,
Calif.) to allow larger treatment spots as desired. Seventeen (17)
areas of choroidal neovascularization were treated using a 1250
.mu.m spot. Treatment spot sizes were confirmed at the treatment
plane using a Dial caliper micrometer. Some areas of choroidal
neovascularization were treated with several adjacent treatment
spots to treat the whole area of choroidal neovascularization. One
large choroidal neovascular membrane was treated with photodynamic
therapy to the nasal half only.
The photodynamic irradiation treatments were carried out with a
plano fundus contact lens (OGFA, Ocular Instruments, Inc., Bellvue,
Mass.). Power was verified at the cornea by a power meter (Coherent
Fieldmaster, Coherent, Auborn, Calif.). The fluence at each
treatment spot was 50, 75, 100 or 150 Joules/cm.sup.2. Initially,
the irradiance was set at 150 mW/cm.sup.2 to avoid any thermal
effect but, as the experiment proceeded, the irradiance was
increased to 300 mW/cm.sup.2 or 600 mW/cm.sup.2 to reduce the
treatment duration time. The time interval between injection of the
green porphyrin dye and the treatment irradiating step ranged from
about 1 to about 81 minutes.
A number of different combinations of parameter values were studied
and are summarized below in Table 1:
TABLE 1 ______________________________________ IRRADIANCE AT 150
mW/cm.sup.2 Dura- Time tion of after Closure Number Dye Treat-
Injec- by of CNV dose Fluence ment tion Angio- Treated (mg/kg)
(J/cm.sup.2) (mins) (mins) graphy
______________________________________ 2 2 50 5.6 18, 38 2/2 1 2 75
8.3 81 1/1 1 2 100 11.2 22 1/1 2 1 50 5.6 5, 30 0/2 3 1 100 11.2 1,
2 3/3 and 5 4 1 150 16.6 14-43 3/4
______________________________________
"Dye only" controls, which were exposed to dye but not to laser
light, were examined in the areas of normal retina/choroid. Areas
of choroidal neovascularization were examined angiographically and
histologically. "Light only" controls were not performed, since the
irradiances used for photodynamic therapy were well below the
levels used for clinical laser photocoagulation. (In a related
experiment, a minimally detectable lesion using "light-only"
required an irradiance of 37 W/cm.sup.2, about 100 times the light
levels used for photodynamic therapy.)
Following photodynamic therapy, the monkeys were returned to an
animal care facility. No attempt was made to occlude the animals'
eyes, but the room in which they were housed was darkened
overnight.
The condition of the choroidal neovasculature was followed by
fundus photography, fluorescein angiography, and histologic
examination. In particular, the eyes of the monkeys were examined
by fluorescein angiography acutely and at 24 hours after the
photodynamic therapy was given. In some cases, follow-up by
fluorescein angiography was performed at 48 hours and at one week,
until the eyes were harvested and the animals killed at the
following time points: acutely, at 24 hours, 48 hours, and 8 days
following photodynamic therapy. Animals were sacrificed with an
intravenous injection of 25 mg/mg Nembutal.
To perform the histologic examination, all eyes were enucleated
under deep anesthesia and fixed overnight in modified Karnovsky's
fixative, and then transferred to 0.1M phosphate buffer, pH 7.2 at
4.degree. C. Both light microscopy and electron microscopy were
used for these studies. For light microscopy, tissue samples were
dehydrated, embedded in epon and serially sectioned at one micron.
The sections were stained with tolnizin blue and examined with an
Olympus photomicroscope. For electron microscopy, tissue samples
were post-fixed in 2% osmium tetroxide and dehydrated in ethanol.
Sections were stained with uranyl acetate in methanol, stained with
Sato's lead stain, and examined with a Philips #CM 10 transmission
electron microscope.
Using the low irradiance level of 150 mW/cm.sup.2 to minimize any
thermal component of the treatment, green porphyrin doses of 1-2
mg/kg of BPD-MA/LDL, and fluences of 50-150 Joules/cm.sup.2,
choroidal neovascularization was effectively closed. Using the
higher 2 mg/kg dose effectively closed choroidal
neovascularizations at even the lowest 50 Joules/cm.sup.2 fluence.
When the green porphyrin dose was decreased to 1 mg/kg of
BPD-MA/LDL to minimize damage to surrounding tissues, the fluence
required to effectively close choroidal neovascular tissue
increased to 100 Joules/cm.sup.2. At 100 and 150 Joules/cm.sup.2,
the treated choroidal neovascular tissue was angiographically
closed, as shown by hypofluorescence in the area of treatment.
Prior to photodynamic therapy, the areas of choroidal
neovascularization exhibited a gray sub-retinal elevation that
leaked profusely on fluorescein angiography. There was no apparent
color change in the treated areas either during or immediately
after photodynamic treatment. However, 24 hours after the
irradiating step, there was mild retinal whitening in the treated
areas.
Further fluorescein angiography showed hypofluorescence in the
treated areas, with no apparent filling of the associated
neovascular tissues. Retinal vessels within the treated areas were
perfused, but stained later. A hyperfluorescent rim at the border
of the treated area was apparent in the later frames of the
angiograph, and the rim then progressed to fill the treated area.
Although mild staining of retinal vessels was noted
angiographically, no complications, such as serous retinal
detachment or hemorrhage, were noted.
On histopathologic examination of the 2 mg/kg dose samples, there
was marked disruption of the treated choroidal neovascular tissue
with disrupted endothelial cells. The choriocapillaris was also
occluded. Although large choroidal vessels were unaffected,
extravasated red blood cells were noted in the choroid. Retinal
pigment epithelium (RPE) damage was noted as well with vacuolated
cells, with the outer nuclear layer demonstrating pyknotic nuclei
and disrupted architecture. No histologic abnormality of the
retinal vessels was seen.
Histolopathologic examination of the 1 mg/kg dose samples showed
damage to endothelial cells in the choroidal neovascular tissue,
with abnormal nuclei and disrupted cytoplasm in the endothelial
cells. The lumens of the vessels in the choroidal neovascular
tissue were occluded by fibrin acutely and were closed by 24 hours
after treatment. Closure of the choroicapillaris was also noted. At
24 hours, the retinal pigment epithelium (RPE) appeared abnormal
with vacuolated cytoplasm. Pyknotic nuclei in the inner and outer
layer indicated damage secondary to the laser injury used to induce
the neovascularization in this model. Retinal vessels appeared to
be undamaged.
Choroidal neovascular tissue that was treated and followed for
eight days showed persistent closure, as shown by hypofluorescence
in the early frames of the angiogram. Histologically, the treated
areas demonstrated degraded vessel lumens empty of debris. The
choriocapillaris was sparse but patent in the treated area. In
contrast, areas of choroidal neovascularization not treated by
photodynamic therapy demonstrated branching capillaries between
Bruch's membrane and the outer retina.
No adverse effects of photodynamic therapy with the green porphyrin
were noted. There was no associated serous retinal detachment,
retinal or sub-retinal hemorrhage, or post-treatment inflammation.
Further, no adverse systemic effects of the dye administration were
noted. However, the low irradiance forced treatment times to be
long--about 16.6 minutes to yield 150 Joules/cm.sup.2.
EXAMPLE 2
Control of Experimental Choroidal Neovascularization Using PDT with
BPD-MA/LDL at Higher Irradiances
To make clinical treatments shorter, additional experiments were
performed using higher irradiance values. Experience with higher
irradiance indicated that no thermal damage would take place with
irradiances as high as 1800 mW/cm.sup.2. Moulton et al., "Response
of Retinal and Choroidal Vessels to Photodynamic Therapy Using
Benzoporphyrin Derivative Monoacid", IOVS 34, 1169 (1993), Abstract
2294-58. Therefore, irradiances of 300 mW/cm.sup.2 and 600
mW/cm.sup.2 were also used to treat choroidal neovascular tissue in
accordance with the procedures described in Example 1. The results
showed that shortened treatment times effectively closed the
choroidal neovascular tissue, as indicated below in Table 2.
TABLE 2 ______________________________________ IRRADIANCE OVER 150
mW/cm.sup.2 Irra- Duration of Time after Closure Number Dye diance
Treat- Injec- by of CNV dose Fluence (mW/ ment tion Angio- Treated
(mg/kg) (J/cm.sup.2) cm.sup.2) (mins) (mins) graphy
______________________________________ 2 1 150 300 8.3 5, 53 2/2 2
1 150 600 4.7 22, 69 2/2 ______________________________________
Occlusion of the choroidal neovascular tissue and subjacent
choriocapillaris was observed, as well as damage to the retinal
pigment epithelium and outer retina.
EXAMPLE 3
Control of Experimental Choroidal Neovascularization Using PDT with
BPD-MA Liposomes
The following experiment of photodynamic therapy using a liposomal
preparation of BPD-MA was conducted to determine the optimal time
interval after intravenous injection as a bolus of the BPD-MA over
about 20 seconds, followed by a 3-5 cc saline flush, to begin the
irradiating step. Choroidal neovascularization in cynomolgus
monkeys was treated to demonstrate efficacy of the photodynamic
therapy. Normal choroid tissue was treated to assess relative
damage to adjacent tissues.
The monkeys were initially injected with a green porphyrin dose of
1 mg/kg. At predetermined time intervals following this injection,
the eyes of the monkeys were irradiated with an irradiance of 600
mW/cm.sup.2, and a fluence of 150 J/cm.sup.2. The irradiating light
was from an argon/dye laser (Coherent 920 Coherent Medical Laser,
Palo Alto, Calif.) equipped with a 200 micron fiber adapted through
a LaserLink (Coherent Medical Laser) and a split lamp delivery
system (Coherent). Other than these differences, the eye membranes
were treated in the same manner as described in Example 1. All
areas of treated choroidal neovasculature for all time points after
the liposomal BPD-MA injection showed whitening of the retina and
early hypofluorescense on fluorescein angiography when measured one
week after treatment. On histology, there was evidence of partial
closure of choroidal neovasculature at the early time points, no
effect at mid-time points, and more effective closure at late
irradiation time points, e.g., at 80 and 100 minutes.
The normal choroid treated with the same parameters showed
whitening of the retina, early hypofluorescence at all time points,
and histologic evidence of choroicapillaris (c-c) accompanied by
damage to the choroid and retina, particularly at early time
points.
EXAMPLE 4
Using PDT with BPD-MA Liposomes at Lower Green Porphyrin Doses
Using the general procedure of Example 1, additional experiments
were performed using the intravenous injection of liposomal BPD-MA
at doses of 0.25, 0.5 and 1 mg/kg. Photodynamic therapy was
performed with an irradiance of 600 mW/cm.sup.2, a fluence of 150
J/cm.sup.2, and a treatment duration of four minutes, nine
seconds.
The effects of treatment were assessed by fundus photography and
fluorescein angiography, and then confirmed by light and electron
microscopy. Photodynamic therapy of normal choroid tissue
demonstrated the effect on adjacent structures, such as the retina,
while the treatment of choroid neovascular tissue demonstrated
efficacy.
Table 3 below describes the lesions produced on normal choroids by
administration of 0.5 mg/kg BPD-MA at time points ranging from 5 to
60 minutes:
TABLE 3 ______________________________________ 0.5 mg/kg, NORMAL
CHOROID Time after injection Fluorescein (min) Angiography
Histology ______________________________________ 5 Hypofluorescence
c-c and large choroidal vessel closure; outer and inner retina
damage. 20 Hypofluorescence; cc closure; damage retinal vessels -
to outer retina normal 40 Mild early cc open (not center
hypofluorescence of lesion); outer retina damage 60 Early cc
closed; outer hypofluorescence; retina damage; less than the 20-
inner retina fairly minute lesion good. described above
______________________________________
When 0.5 mg/kg BPD-MA was also used to treat choroidal
neovasculature under the same conditions, marked hypofluorescence
corresponding to closure of choroid neovasculature was exhibited in
areas irradiated at times of 5, 20 and 40 minutes after injection.
When 50 minutes after injection were allowed to elapse before
photodynamic irradiation was begun, there was less hypofluorescence
and presumably less effective closure.
The study was then repeated with the green porphyrin dose decreased
to 0.25 mg/kg. Table 4 below describes the lesions produced on
normal choroids by treatments with 0.25 mg/kg, 600 mW/cm.sup.2, and
150 J/cm.sup.2 at time points ranging from 5 to 60 minutes:
TABLE 4 ______________________________________ 0.25 mg/kg, NORMAL
CHOROID Time after injection Fluorescein (min) Angiography
Histology ______________________________________ 10 Early c-c
closure; hypofluorescence choroidal vessel - normal; RPE damaged;
retinal vessels - normal; mild damage to outer retina 20 Early Same
as 10-minute hypofluorescence lesion above 40 Faint early Patchy cc
closure; hypofluorescence; less damage to RPE late staining and
outer retina 60 Not demonstrated No effect on cc; mild
vacuolization of RPE ______________________________________
When the above study was repeated using the same green porphyrin
dose of 0.25 mg/kg and irradiance of 600 mW/cm.sup.2, but with a
reduced fluence of 100 J/cm.sup.2, the same angiographic and
histologic pattern was exhibited as described above. However, cc
was open in the 40-minute lesion.
In the last portion of these experiments, a green porphyrin dose of
0.25 mg/kg was used to treat experimental choroidal
neovascularization with an irradiance of 600 mW/cm.sup.2 and a
fluence of 150 J/cm.sup.2 at elapsed time points ranging from 5 to
100 minutes. This combination of conditions caused effective cc
closure with only minimal damage to the outer retina. The results
are shown in Table 5 below:
TABLE 5 ______________________________________ 0.25 mg/kg, PDT over
CNV Time after injection Fluorescein (min) Angiography Histology
______________________________________ 5 Early Partially closed
hypofluorescence CNV; c-c closed; damage to inner retina 20 Early
CNV - open vessel, hypofluorescence; fibrin and clots; less than
the 5- inner retina looks minute lesion fine 30 Some Minimal effect
on hypofluorescense CNV next to CNV 40 Hypofluorescence; Minimal
effect on questionable change CNV compared to previous reaction 60
Hypofluorescence Minimal effect on CNV 80 Hypofluorescence Partial
closure of CNV; retina over CNV looks intact 100 Hypofluorescence
CNV partially closed ______________________________________
Thus, fluorescein angiography and histopathology in the above
series of experiments demonstrated early hypofluorescence at early
time points. Further, the histopathology study showed partial CNV
closure at all time points after injection using 80 and 100 minutes
as the post-injection interval before the irradiating
treatment.
In summary, acceptable destruction of choroidal neovascular tissue
at all tested doses of BPD-MA was shown by fluorescein angiography
and histology. However, the lower doses appeared to increase
selectivity, as assessed by treatment of a normal choroid.
Effective choriocapillaris closure in normal choroids with minimal
retinal damage was produced by irradiating at a time about 10
minutes, 20 seconds after injection of the green porphyrin at a
dose of 0.25 mg/kg. By adjusting the dose, the time of irradiation
after green porphyrin injection, and fluence, one can improve even
further the selectivity of the green porphyrin. However, the
liposomal preparation of BPD-MA was clearly demonstrated to be a
potent photosensitizer.
* * * * *